Synthesis, DNA binding, cytotoxicity and sequence specificity of a series of imidazole-containing analogs of the benzoic acid mustard distamycin derivative tallimustine containing an alkylating group at the C-terminus

In an attempt to produce additional alkylation and crosslinking in the minor groove of DNA, imidazole-containing analogs of distamycin were synthesized with benzoic acid mustard (BAM) and methoxyaziridinyl moieties present at the N- and C-termini, respectively. Analogs 1a-c differed in the number of methylene units (2-4 respectively) between the C-terminal carbonyl group and the methoxyaziridinyl moiety. DNA binding affinity to several polynucleotides decreased with increasing linker length, whereas DNA interstrand crosslinking ability, as measured by a plasmid gel based assay, increased. The in vitro cytotoxicity in human chronic myeloid leukemia K562 cells and the panel of human tumor cell lines at the National Cancer Institute decreased with increasing number of methylene units, and no increase in cytotoxicity was observed over compound AR-1-122 which did not contain the methoxyaziridinyl moiety. 1a-c had the same sequence selectivity of alkylation as AR-1-122, showing alkylation only at 5'-TTTTGPu sequences. The relative binding to these sequences decreased with increasing number of methylene units. The addition of a methoxyaziridinyl moiety in this group of imidazole and BAM-containing compounds can, therefore, increase crosslinking ability to naked DNA but this does not result in an increase in cytotoxicity. In contrast the cytotoxicity was related to their ability to produce sequence specific alkylation at 5'-TTTTGPu sequences.     

DNA alkylation and interstrand cross-linking by treosulfan

The anti-tumour drug treosulfan (L-threitol 1,4-bismethanesulphonate, Ovastat) is a prodrug for epoxy compounds by converting non-enzymatically to L-diepoxybutane via the corresponding monoepoxide under physiological conditions. The present study supports the hypothesis that this conversion of treosulfan is required for cytotoxicity in vitro. DNA alkylation and interstrand cross-linking of plasmid DNA is observed after treosulfan treatment, but this is again produced via the epoxide species. Alkylation occurs at guanine bases with a sequence selectivity similar to other alkylating agents such as the nitrogen mustards. In treosulfan-treated K562 cells, cross-links form slowly, reaching a peak at approximately 24 h. Incubation of K562 cells with preformed epoxides shows faster and more efficient DNA cross-linking.     

DNA-Directed alkylating agents. 7. Synthesis, DNA interaction, and antitumor activity of bis(hydroxymethyl)- and bis(carbamate)-substituted pyrrolizines and imidazoles

A series of bis(hydroxymethyl)-substituted imidazoles, thioimidazoles, and pyrrolizines and related bis(carbamates), linked to either 9-anilinoacridine (intercalating) or 4-(4-quinolinylamino)benzamide (minor groove binding) carriers, were synthesized and evaluated for sequence-specific DNA alkylation and cytotoxicity. The imidazole and thioimidazole analogues were prepared by initial synthesis of [(4-aminophenyl)alkyl]imidazole-, thioimidazole-, or pyrrolizine dicarboxylates, coupling of these with the desired carrier, and reduction to give the required bis(hydroxymethyl) alkylating moiety. The pyrrolizines were the most reactive alkylators, followed by the thioimidazoles, while the imidazoles were unreactive. The pyrrolizines and some of the thioimidazoles cross-linked DNA, as measured by agarose gel electrophoresis. Strand cleavage assays showed that none of the compounds reacted at purine N7 or N3 sites in the gpt region of the plasmid gpt2Eco, but the polymerase stop assay showed patterns of G-alkylation in C-rich regions. The corresponding thioimidazole bis(carbamates) were more selective than the bis(hydroxymethyl) pyrrolizines, with high-intensity bands at 5'-NCCN, 5'-NGCN and 5'-NCGN sequences in the PCR stopping assay ( indicates block sites). The data suggest that these targeted compounds, like the known thioimidazole bis(carbamate) carmethizole, alkylate exclusively at guanine residues via the 2-amino group, with little or no alkylation at N3 and N7 guanine or adenine sites. The cytotoxicities of the compounds correlated broadly with their reactivities, with the bis(hydroxymethyl)imidazoles being the least cytotoxic (IC50s >1 microM; P388 leukemia) and with the intercalator-linked analogues being more cytotoxic than the corresponding minor-groove-targeted ones. This was true also for the more reactive thioimidazole bis(carbamates) (IC50s 0.8 and 11 microM, respectively), but both were more active than the analogous "untargeted" carmethizole (IC50 20 microM). The bis(hydroxymethyl)pyrrolizine analogues were the most cytotoxic, with IC50s as low as 0.03 microM.     

Enzymatic and chemical footprinting of anthracycline antitumor antibiotics and related saccharide side chains

DNase I and three DNA chemical footprinting agents were used to compare the DNA binding properties of the anthracycline antitumor antibiotics daunomycin, aclacinomycin A, and ditrisarubicin B. These anthracyclines contain a tetracyclic chromophore which intercalates into DNA and a monosaccharide, trisaccharide, and two trisaccharide side chains, respectively. These side chains consist of between one and three 2,6-dideoxy, 1,4-diaxially linked sugars. Three chemical probes, fotemustine, dimethyl sulfate, 4-(2'-bromoethyl)phenol, and the enzymic probe DNase I were used in the footprinting experiments. The chemical probes provided a clear picture of the binding pattern at 37 degrees C and more detailed information than that obtained using the standard DNase I footprinting assay. All three anthracyclines showed preferred binding to 5'-GT-3' sequences in both the chemical and enzymatic footprinting. DNase I footprinting showed that the number of base pairs of DNA protected from cleavage increased with the number of saccharide groups present at particular sites and is consistent with DNA binding of the saccharide side chains. Alkylation of runs of guanine by fotemustine was inhibited by all three anthracyclines, while alkylation by dimethyl sulfate was enhanced for most guanines. The probe 4-(2'-bromoethyl)phenol showed that all three anthracyclines completely protected all of the adenines in the minor groove from alkylation, and enhanced major groove guanine alkylation was observed with aclacinomycin A, daunomycin, and, to a much lesser extent, ditrisarubicin B. These results are consistent with intercalation of the aglycone ring and binding of the rigid, hydrophobic saccharide side chains in the minor groove. Footprinting of four methyl glycosides related to the anthracyclines showed no evidence of DNA binding with any of the agents studied.     

Molecular dynamics simulations of chlorambucil/DNA adducts. A structural basis for the 5'-GNC interstrand DNA crosslink formed by nitrogen mustards

The alkylation of DNA by chlorambucil has been studied using a computational approach. Molecular dynamics simulations were performed on the fully solvated non-covalent complex, two monoadducts and a crosslinked diadduct of chlorambucil with the d(CGG3G2CGC).-d(GCG1CCCG) duplex, in which the N7 atoms of G1, G2 and G3 are potential alkylation sites. The results provide a structural basis for the preference of nitrogen mustards to crosslink DNA duplexes at a 5'-GNC site (a 1,3 crosslink, G1-G3) rather than at a 5'-GC sites (a 1,2 crosslink, G1-G2). In the non-covalent complex simulation the drug reoriented from a non-interstrand crosslinking location to a position favorable for G1-G3 diadduct formation. It proved possible to construct a G1-G3 diadduct from a structure from the non-covalent simulation, and continue the molecular dynamics calculation without further disruption of the DNA structure. A crosslinked diadduct developed with four BII conformations on the 3' side of each alkylated guanine and of their respective complementary cytosine. In the first monoadduct simulation the starting point was the same DNA conformation used in the crosslinked diadduct simulation with alkylation at G1. In this simulation the DNA deformation was reduced, with the helix returning to a more canonical form. A second monoadduct simulation was started from a canonical DNA conformation alkylated at G3. Here, no significant motion towards a potential crosslinking conformation occurred. Collectively, the results suggest that crosslink formation is dependent upon the drug orientation prior to alkylation and the required deformation of the DNA to permit 1,3 crosslinking can largely be achieved in the non-covalent complex.     

DNA minor groove binding-directed poisoning of human DNA topoisomerase I by terbenzimidazoles

We have employed a broad range of spectroscopic, calorimetric, DNA cleavage, and DNA winding/unwinding measurements to characterize the DNA binding and topoisomerase I (TOP1) poisoning properties of three terbenzimidazole analogues, 5-phenylterbenzimidazole (5PTB), terbenzimidazole (TB), and 5-(naphthyl[2,3-d]imidazo-2-yl)bibenzimidazole (5NIBB), which differ with respect to the substitutions at their C5 and/or C6 positions. Our results reveal the following significant features. (i) The overall extent to which the three terbenzimidazole analogues poison human TOP1 follows the hierarchy 5PTB > TB > 5NIBB. (ii) The impact of the three terbenzimidazole analogues on the superhelical state of plasmid DNA depends on the [total ligand] to [base pair] ratio (rbp), having no effect on DNA superhelicity at rbp ratios < or = 0.1, while weakly unwinding DNA at rbp ratios > 0.1. This weak DNA unwinding activity exhibited by the three terbenzimidazoles does not appear to be correlated with the abilities of these compounds to poison TOP1. (iii) Upon complexation with both poly(dA).poly(dT) and salmon testes DNA, the three terbenzimidazole analogues exhibit flow linear dichroism properties characteristic of a minor groove-directed mode of binding to these host DNA duplexes. (iv) The apparent minor groove binding affinities of the three terbenzimidazole analogues for the d(GA4T4C)2 duplex follow a qualitatively similar hierarchy to that noted above for ligand-induced poisoning of human TOP1-namely, 5PTB > TB > 5NIBB. In the aggregate, our results suggest that DNA minor groove binding, but not DNA unwinding, is important in the poisoning of TOP1 by terbenzimidazoles.     

Mustard prodrugs for activation by Escherichia coli nitroreductase in gene-directed enzyme prodrug therapy

Twenty nitrogen mustard analogues derived from 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954, 1) were evaluated as candidate prodrugs for gene-directed enzyme prodrug therapy (GDEPT) in Chinese hamster V79 cell lines engineered to express Escherichia coli nitroreductase (NR). Structural variations within the series included the use of N-dihydroxypropyl and (N-dimethylamino)ethyl carboxamide side chains, the use of chloro, bromo, mesyl, and iodo leaving groups on the mustards, and regioisomeric changes. The compounds were assayed for cytotoxicity (IC50) with the NR-expressing and controls of non-NR-expressing cell lines. The proportion of NR-expressing cells required in a mixture for nonexpressing cells to experience 50% of their cytotoxicity (termed the TE50) was used to assess the compounds' ability to induce a bystander effect. This study suggests that 5-[N,N-bis(2-bromoethyl)amino]-2,4-dinitrobenzamide (8), 5-[N,N-bis(2-iodoethyl)amino]-2,4-dinitrobenzamide (9), 2-[N,N-bis(2-bromoethyl)-amino]-3,5-dinitrobenzamide (13), and 2-[N,N-bis(2-iodoethyl)amino]-3,5-dinitrobenzamide (14) showed considerable improvements over 1, exhibiting greater potency, higher IC50 ratios, and lower TE50s, and are thus superior prodrugs to 1 for GDEPT.     

Single chain dimers of MASH-1 bind DNA with enhanced affinity

By designing recombinant genes containing tandem copies of the coding region of the BHLH domain of MASH-1 (MASH-BHLH) with intervening DNA sequences encoding linker sequences of 8 or 17 amino acids, the two subunits of the MASH dimer have been connected to form the single chain dimers MM8 and MM17. Despite the long and flexible linkers which connect the C-terminus of the first BHLH subunit to the N-terminus of the second, a distance of approximately 55 A, the single chain dimers could be produced in Escherichia coli at high levels. MM8 and MM17 were monomeric and no 'cross-folding' of the subunits was observed. CD spectroscopy revealed that, like wild-type MASH-BHLH, MM8 and MM17 adopt only partly folded structures in the absence of DNA, but undergo a folding transition to a mainly alpha-helical conformation on DNA binding. Titrations by electrophoretic mobility shift assays revealed that the affinity of the single chain dimers for E box-containing DNA sequences was increased approximately 10-fold when compared with wild-type MASH-BHLH. On the other hand, the affinity for heterologous DNA sequences was increased only 5-fold. Therefore, the introduction of the peptide linker led to a 4-fold increase in DNA binding specificity from -0.14 to -0.57 kcal/mol.     

Comparison of the sequence-selective DNA binding by peptide dimers with covalent and noncovalent dimerization domains

Sequence-specific DNA binding proteins generally consist of more than two DNA-contacting regions to ensure the selectivity of recognition. The multiple DNA binding modules are connected either through the covalent linker or through the noncovalent dimerization domain. We have compared the DNA binding of peptide dimers with covalent and noncovalent dimerization domains to explore the potential advantage of each linkage on the sequence-specific DNA binding. Three sets of head-to-tail peptide dimers were synthesized by using the same basic region peptide to target the same DNA sequence; one dimer was assembled with a bridged biphenyl derivative as a covalent dimerization domain, and two other dimers were assembled with the cyclodextrin guest noncovalent dimerization domains. One of the noncovalent dimers was a heterodimer that consisted of cyclodextrin and guest peptides, while the other was a homodimer that consisted of peptides bearing both cyclodextrin and the guest molecule within the same chain. Both noncovalent dimers formed the specific DNA complexes within narrower ranges of peptide concentrations and showed higher sequence selectivity than the covalent dimer did. Among the three dimers, the noncovalent homodimer that can form an intramolecular inclusion complex showed the highest sequence selectivity. Because the noncovalent homodimer with the higher stability of the circular intramolecular inclusion complex exhibited the higher sequence selectivity, it was concluded that an equilibrium involving a conformational transition of a monomeric peptide effectively reduced the stability of its nonspecific binding complex, hence increasing the efficacy of cooperative dimer formation at the specific DNA sequence.     

Combination of the new minor groove alkylator tallimustine and melphalan

The benzoyl nitrogen mustard derivative of distamycin A, tallimustine, belongs to a new class of alkylating agents, known as DNA minor groove alkylating agents. It alkylates adenine N3 with high sequence specificity, causing no alkylation of guanine N7, the main site of alkylation of clinically used nitrogen mustards such as L-PAM. The present study investigated the in vivo antitumour activity of a combination of tallimustine and melphalan (L-PAM). Two murine tumours were used: i.p. (intraperitoneally) transplanted L1210 leukaemia and i.m. (intramuscularly) transplanted M5076 ovarian reticulum cell sarcoma (M5). In L1210, which is only marginally sensitive to tallimustine, the combination of tallimustine 3 mg/kg i.p. with L-PAM 10 mg/kg i.p. was as effective as 20 mg/kg L-PAM, which is the maximum tolerated dose. In M5, which is sensitive to both drugs, the combination was superior to either drug alone. The results suggest that the combination of tallimustine and L-PAM--or possibly in general, minor groove alkylators and major groove alkylators--may be therapeutically advantageous and therefore should be investigated clinically.     

Differences in DNA recognition and conformational change activity between boxes A and B in HMG2 protein

High mobility group (HMG) 2 is a sequence-nonspecific DNA-binding protein consisting of a repeat of DNA-binding domains called HMG1/2 boxes A and B and an acidic C-terminal. To understand the mode of HMG2 interaction with DNA, we expressed various HMG2 peptides containing HMG1/2 box(es) in Escherichia coli cells and purified them. Gel retardation and DNA supercoiling assay indicated that the region essential for the preferential binding of HMG2 with negatively supercoiled DNA and DNA unwinding activity is located in box B, but not sufficient alone. The flanking C-terminal basic region or box A linked by a linker region is necessary to express activities. The SPR measurements certified that the intrinsic DNA binding affinity of box B is weaker (Kd = 170 microM), and these adjoining regions largely strengthen the affinity (Kd 相似文献   

Detection of the sites of alkylation in DNA and polynucleotides by laser Raman spectroscopy

A laser Raman study of the alkylation of calf thymus DNA, poly(dG)-poly(dC) and poly(dA)-(dT) has been made using two water soluble alkylating agents: an antitumor drug, the difunctional methyl nitrogen mustard (HN2), which froms interstrand cross-links, and the dimethyl nitrogen half mustard (HN1). When an excess of the alkylating agent was used, the observed Raman frequencies due to the guanine ring modes in DNA and poly(dG)-poly(dC) changed virtually quantitatively to those of 7-methylguanosine (7-Me-Guo) showing that essentially all of the guanine bases were alkylated in the N-7 position. Furthermore, this alkylated DNA formed a stable double helical complex at neutral pH in which the alkylated guanine residues are in the keto form. No changes in the Raman bands of any of the other bases were observed in alkylated DNA. The DNA double helix, completely alkylated in at the N-7 position of guanine, melts about 35 degrees C below that of the native DNA. Upon melting, the alkylated guanine changes from the keto to the zwitterionic form.     

The binding of ethyl carbamate to DNA of mouse liver in vivo: the nature of the bound molecule and the site of binding

Ethyl carbamate, labelled at C1 with 14C, bound in vivo to liver DNA of intact and partially hepatectomised mice. Isotope (18O) enrichment was not detected in the oxygen of liver DNA of mice injected with [18O] ethyl carbamate, C2H5--18O--CO--NH2. This suggests that it was the ethyl group and not the ethoxy group which bound to DNA. Chromatographic analysis of acid hydrolysates of liver DNA from mice treated with [1-14C] ethyl carbamate provided no evidence of alkylation or other form of binding to purine or pyrimidine bases. On relatively mild acid hydrolysis the alkyl group remained bound to the "apurinic acid" fraction, while more vigorous hydrolysis lead progressively first to its separation as highly ionisable hydrophilic non-volatile compounds and then to its loss as a volatile compound. DNAase I followed by phosphodiesterase hydrolysis also split off the 14C-containing group as a volatile compound. The volatile compound was identified as ethanol. These results suggest that the alkyl group was bound as an ester to a phosphate group in the DNA chain. Results with DNA from partially hepatectomised mice did not differ from those with DNA from intact mice.     

Synthesis of new bifunctional compounds which selectively alkylate guanines in DNA

The aim of this work was to develop new bifunctional alkylating agents which damage DNA in a selective manner. In order to extend our previously published work on conformationally restricted nitrogen mustards containing one piperidine ring, new bispiperidine derivatives were designed with varying lengths of carbon chain between the two rings and structure-activity relationships in these systems were studied. Thus samples of new bispiperidine salts 22-26 with chloromethyl groups at the 2-positions and a bridge between the two nitrogen atoms of 2-6 carbon atoms were synthesized. The analogous new bis(p-nitrophenylcarbamates) 17-21 were also prepared. The free bases were designed to be bifunctional alkylating agents via aziridinium ion formation with different distances between the two alkylating sites. The bispiperidines 22-24 were shown to alkylate guanines at the 7-position in the major groove of DNA more selectively than melphalan. The bispiperidine 22 with the shortest two carbon bridge was the most reactive but it was less cytotoxic than melphalan in a human colon carcinoma cell line (IC50 value approximately 30 microM) and in a human chronic myeloid leukaemia cell line (IC50 value approximately 12 microM). The most cytotoxic compound in the latter cell line was the carbamate 17, with an IC50 value of approximately 0.3 microM, and carbamates 17, 19 and 20 were most potent in a panel of human ovarian carcinoma cell lines. These compounds also showed circumvention of acquired cisplatin resistance in three paired cell lines. The carbamates 17-21, however, were less efficient at alkylating and cross-linking naked DNA than the bispiperidines 22-26.     

Peptide nucleic acids and their phosphonate analogues: synthesis and hybridization characteristics

The synthesis of a series of DNA mimics--peptide nucleic acids, phosphonate analogues of peptide nucleic acids, and their hybrids--is described. The preparative synthesis of the corresponding monomers and the solid phase automated synthesis of oligomers-mimics are developed. Modified phosphonate analogues of peptide nucleic acids, in particular chiral derivatives and those with additional hydroxyl groups in the side chains of the backbone as well as pyrene derivatives of peptide nucleic acids and their phosphonate analogues, are prepared. The ability of the resulting oligomers specifically to hybridize to DNA and RNA complementary chains is studied. It is shown that phosphonate analogues of peptide nucleic acids and their hybrids with peptide nucleic acids can form complexes with the DNA and RNA complementary strands, the stability of the complexes increasing in parallel with the increase in the number of peptide nucleic acid residues in the chain of the mimic. This property, along with good water solubility, provides the precondition for further evaluation of these compounds as antisense and antigene agents.     

Restricted usage of T cell receptor V alpha/J alpha gene segments with different nucleotide but identical amino acid sequences in HLA-DR3+ sarcoidosis patients

Alkylation of DNA was studied after treatment with [3H]melphalan (phenylalanine mustard; 1-2 microM) using a human tumour cell line, RD, in culture, or Escherichia coli (WP2 or WP2-uvrA strains) in growth medium. After 6 h at 37 degrees C, treated cells were isolated and re-suspended in fresh growth media. Samples were taken at times up to 48 h for isolation of DNA, and in some cases also RNA and protein (which were found to be alkylated to about the same extent as DNA). Alkylated DNA was analysed as previously described (M.R. Osborne and P.D. Lawley, Chem.-Biol. Interact 89 (1993) 49-60). The four principal products, mono-7-alkylguanine (G-M-OH); mono-3-alkyladenine (A-M-OH); and the cross-linked products G-M-G and A-M-G, were identified in DNA from melphalan treated cells, and quantitatively determined. In each case, alkylation of cellular macromolecules was maximal after about 6 h. In DNA of the human tumour cell line, the relative amounts of adenine products decreased with time, most markedly with A-M-OH to 42% of the 2-h value after 48 h. In DNA of both bacterial strains, A-M-OH was virtually undetectable even at early times. Comparisons between the time course of relative decreases in amounts of these alkylpurine products and the corresponding values for alkylated DNA in vitro suggest that the adenine products are subject to removal by repair enzyme action in E. coli of either strain.     

GC base sequence recognition by oligo(imidazolecarboxamide) and C-terminus-modified analogues of distamycin deduced from circular dichroism, proton nuclear magnetic resonance, and methidiumpropylethylenediaminetetraacetate-iron(II) footprinting studies

The DNA binding properties of a series of imidazole-containing and C-terminus-modified analogues 4-7 of distamycin are described. These analogues contain one to four imidazole units, respectively. Data from the ethidium displacement assay showed that these compounds bind in the minor groove of DNA, with the relative order of binding constants of 6 (Im3) > 7 (Im4) > 5 (Im2) > 4 (Im1). The reduced binding constants of these compounds for poly(dA-dT) relative to distamycin, while they still interact strongly with poly(dG-dC), provided evidence of GC sequence acceptance. The preferences for GC-rich sequences by these compounds were established from a combination of circular dichroism (CD) titration, proton nuclear magnetic resonance (1H-NMR), and methidiumpropylethylenediaminetetraacetate-iron(II) [MPE.Fe-(II)] footprinting studies. In the CD studies, these compounds produced significantly larger DNA-induced ligand bands with poly(dG-dC) than poly(dA-dT) at comparable ligand concentrations. 1H-NMR studies of the binding of 5 to d-[CATGGCCATG]2 provided further evidence of the recognition of GC sequences by these compounds, and suggested that the ligand was located on the underlined sequence in the minor groove with the C-terminus oriented over the T residue. MPE footprinting studies on a GC-rich BamHI/SalI fragment of pBR322 provided unambiguous evidence for the GC sequence selectivity for some of these compounds. Compounds 4 and 7 produced poor footprints on the gels; however, analogues 5 and 6 gave strong footprints.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antitumour polycyclic acridines. Part 4. Physico-chemical studies on the interactions between DNA and novel tetracyclic acridine derivatives

The non-covalent interactions between a series of new tetracyclic acridine derivatives (5-11) and DNA have been studied by spectrophotometric analysis, fluorescences quenching, thermal denaturation, and circular and linear dichroism. In order to compare the extent of the DNA binding by compounds 5-11 in their neutral and cationic forms, all experiments were conducted at pH 7.4 (physiological pH) and 5.0. The results indicated that compounds 5-11 are strong DNA-binding ligands with DNA affinities comparable to that of m-AMSA (1) or even higher. They showed a stronger DNA binding activity at pH 5.0 as a result of the N-protonation of the pyridoacridine aromatic chromophore. Ethidium-DNA fluorescence assays showed an A-T base pair preference of the binding distinguishing these novel compounds from simple acridines which show a slight G-C base pair preference. Circular and linear dichroism studies indicated that the drugs bind to DNA by undergoing intercalation inside the duplex macromolecule at high DNA:drug ratios and revealed alternative binding modes at low DNA:drug ratios.     

Investigation of the N-substituent conformation governing potency and mu receptor subtype-selectivity in (+)-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine opioid antagonists

A study of the binding site requirements associated with the N-substituent of (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) derivatives was undertaken using a set of rigid vs flexible N-substituents. The study showed that compounds 7-9 bearing the trans-cinnamyl N-substituent most closely reproduced the potency at the opioid receptor of the flexible N-propylphenyl or N-propylcyclohexyl analogues previously reported. Neither the N-substituted cis-cinnamyl nor the cis-phenylcyclopropylmethyl compounds 10 and 11, respectively, showed high affinity for the opioid receptor. However, the N-trans-phenylcyclopropylmethyl compound 12 closely approximated the affinity of compounds 7-9. Additionally, we found that free rotation of the phenyl ring is necessary for high affinity binding and mu receptor subtype selectivity as the planar N-substituted thianaphthylmethyl and benzofuranylmethyl compounds 13 and 14 had significantly lower binding affinities. Altogether, these findings suggest that the high binding affinity, selectivity, and antagonist potency of N-propylphenyl or N-propylcyclohexyl analogues of (+)-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) are achieved via a conformation wherein the connecting chain of the N-substituents is extended away from piperidine nitrogen with the appended ring system rotated out-of-plane relative to the connecting chain atoms. This conformation is quite similar to that observed in the solid state for 5, as determined by single crystal X-ray analysis. Additionally, it was found that, unlike naltrexone, N-substituents bearing secondary carbons attached directly to the piperidine nitrogen of 4 suffer dramatic losses of potency vs analogues not substituted in this manner. Using a functional assay which measured stimulation or inhibition of [35S]GTP-gamma-S binding, we show that the trans-cinnamyl analogues of (+)-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) retain opioid pure antagonist activity and possess picomolar antagonist potency at the mu receptor.     

Search for DNA repair inhibitors: selective binding of nucleic bases-acridine conjugates to a DNA duplex containing an abasic site

The abasic site is one of the most frequent DNA lesions generated by spontaneous or enzymatic cleavage of the N-glycosidic bond. The abasic site is also an intermediate in the nucleotide and base excision DNA repair. We examined molecules which recognize and cleave DNA at the abasic site with high efficiency. These molecules incorporate in their structure a nucleic base for abasic site recognition, an intercalator for DNA binding, and a polyamino linker for ionic interaction and DNA cleavage. Such compounds, by interfering with abasic sites in DNA, are also inhibitors of DNA repair. In order to better understand the parameters of the interaction, we carried out a UV thermal denaturation study of synthetic oligonucleotides containing the lesion both in the absence and in the presence of the drugs. A similar study was also carried out using the corresponding nonmodified oligonucleotide. The results indicate selective binding of the base-chain-intercalator conjugates to the abasic site containing oligonucleotides.